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LABORATORY DIAGNOSIS OF

PARASITIC DISEASES
OUTLINE
– Macroscopic examination

– Microscopic diagnosis

– Immunological diagnosis

– Molecular diagnosis

– Other techniques

– Specimens for parasitological diagnosis


Learning objective
Upon completion of this unit of instruction and lecture,
the student will be able to:
• List the common techniques used for the detection of
parasites in fecal and blood specimens.
• Discuss the importance of microscope calibration in
parasitology.
• List the types of microscopic methods used in
parasitological investigations.
• Describe the difference between immunodiagnostic
techniques and detection of antigens in parasitology
specimens.
• Discuss the significance of molecular diagnosis in the
diagnosis parasitic diseases.
Type of specimen
Fecal (stool) specimens
Examination of stool
• Purpose of examining stool:
– To identify intestinal parasitic infection associated
» Severe anemia especially in pregnant & child
» Series ill-health
» Persistent diarrhea
» Weight loss, malabsorbtion
» Impairment of development etc

– To identify chronic infection with serious complication


if untreated
– To identify parasitic causes of blood and mucus
– To assist in surveillance &control of parasitic infection
Biosafety
• potential risks with stool specimens includes:
– ingestion of eggs or cysts,
– skin penetration by infective larvae, and
– infection by non-parasitic agents found in stool
and biologic fluids.
• These risks can be minimized:
– by adopting universal precautions as well as
– standard microbiological laboratory practices
(Biosafety Level 2).
Collection of fecal specimens
• recovery and ID depends on:
– proper collection and fixation
– Proper container
• clean, dry, water-proof, wide-mouth container
– Sufficient amount
• About 20-40 grams of well-formed stool or 5-6 table spoonfuls of
watery stool (10ml)
Prequation
• NO contamination with H2O - could bring
free-living protozoans
• NO contamination with urine - destroys trophozoites
• Collect before barium enema - obscures organisms
(7+ days)
• Collect before antibiotics - may decrease number of
organisms
• Series of 3 specimens; 2 normal, 1 purged (if
necessary)
– within 10 days
– on alternate days
Stool: Color
• Normal:
– Adult: brown
– New born infants:- Black(meconium)
– Breast feed infants:- scrambled egg
– Infant feed on animal milk:- “ curd like”
Meconium: Baby’s first stool

• First stool a baby will


pass thick
• Green, tar-like
substance
• Lines the intestines of
the fetus
• First bowel movement
within a few hours after
birth.
Transitional Stool - Stage One
• Newborn slowly
begins to pass the
meconium after birth
• Meconium will begin
to change in
consistency
• Slightly lighter in color
than meconium.
Transitional Stool - Stage Two
• Stool is lighter in color
and slightly less thick
than meconium.
Transitional Stool - Stage Three
• Much lighter and
thinner than
meconium.
• Occurs just before
regular stooling begins
Breastfed Stool
• Yellow
• Runny
• Small seed like objects
in the stool
• Often called baby
poop mustard
Stool: Color
– Changes in the color, consistency, and frequency
of bowel movements is known as a "change in
bowel habits.
– In some cases, an unusual stool color is harmless
and can be attributed to a particular food or
medication
– Changes in stool color that persist can be a serious
matter and should always be investigated by a
physician.
When should you worry about the color of your stool?

• Abnormal:

– Clay or white:
• Absence of bile pigment (bile obstruction) or
• diagnostic study using barium
– Black or tarry:
• Drug (e.g., iron),
• bleeding from upper gastrointestinal tract (e.g.,
stomach, small intestine),
• diet high in red meat and
• dark green vegetables (e.g., spinach)
Stool: Color
• Abnormal:
– Red:
• Bleeding from lower gastrointestinal tract (e.g., rectum),
• hemorrhoids
• some foods
– red gelatin,
– tomato juice or soup

– Pale:
• Malabsorption of fats,
• diet high in milk and milk products and low in meat
B- Consistency of stool
• Varies due to diet but provides information on
the stage of protozoa that is present
• 1-Hard- resists puncture
• 2-formed- can be punctured
• 3- Soft-can be cut with applicator
• 4- Mushy- can be reshaped
• 5- loose- shaped in to container
• 6- Diarrhea- can flows
• 7-Watery- can pour
Stool: Consistency
• Normal: Formed, soft, semisolid or mushy
• Abnormal:
– Hard, dry, constipated stool
• Dehydration, decreased intestinal motility resulting from lack of
fiber in diet, lack of exercise, emotional upset, laxative abuse
– Diarrhea
• Increased intestinal motility (e.g., irritation of the colon by
bacteria) Normal stool is formed, soft, or mushy in consistency
– Cleary watery, loose mixed with mucus and blood
Bristol stool form scale
• Classification of the form,( appearance in a toilet) of
faeces into seven groups.
• The form of the stool depends on the time it spends in
the colon.
• Chart breakdown
– Types 1 and 2 indicate constipation;
– Types 3 and 4 are usually the most comfortable to
pass,
– Types 5-6 tend to be associated with urgency, while
– Type 7 is diarrhea.
Stool: Shape
• Normal: Cylindrical (contour of rectum) about
2.5 cm (1 inch) in diameter in adults

• Abnormal: Narrow, pencil-shaped, or


stringlike stool
– Obstructive conditional of the rectum
Stool: Amount and size
• Normal:
– Amount
• Varies with diet
• About 100 to 400 g per day
– Size
• A healthy piece of feces is about one foot long.
• Shorter sized feces suggest that the colon is not able to
process the food correctly and
• the feces produced does not have the correct amount
of moisture in it.
Stool: Frequency
• HOW OFTEN DOES THE AVERAGE PERSON POOP?
• Normal range
– three times a day to once every three days.
– average person poops about once a day.
• Abnormal
– four times a day or more and
• the stool has a liquid consistency – diarrhea
– less than two or three days a week and
• the stool is hard, dry, and difficult to pass-constipation
Stool: Odor
• WHAT MAKES FECES SMELL SO BAD?
– The distinctive odor of feces is due to bacterial action.
– Specifically, the bacteria produce various compounds
and gases that lead to the infamous smell of feces.
– Gut flora produce compounds such as indole, skatole,
and thiols (sulfur containing compounds), as well as
the inorganic gas hydrogen sulfide
– The bad smell of feces will usually be reduced by
eating more natural foods that do not contain any
artificial flavors or chemicals
Stool: Odor
• Normal: Aromatic, affected by ingested food
and person’s own bacterial flora

• Abnormal: Pungent
– Infection, blood
Stool: Constituents
• Normal:
– water (about 75%).
– Rest constitute:
• dead bacteria that helped us digest our food, living bacteria,
• undigested food residue (known as fiber),
• cellular linings, sloughed epithelial cells
• substances released from the intestines (such as mucus) and the
liver.
• fat, protein, dried constituents of digestive juices (e.g., bile
pigments),
• inorganic matter (e.g., calcium, phosphates)
Stool: Constituents
• Abnormal:
– Pus: bacterial infection
– Mucus: inflammatory condition
– Parasites
– Blood: gastrointestinal bleeding
– Large quantities of fat: malabsorption
– Foreign objects: accidental ingestion
Microscopic examination
• Direct smear
• Smear after concentration
• Permanent stained smear
Preservation
• Required if not delivered to lab immediately
• Preserve protozoan morphology
• Prevents development of worm eggs/larvae
• Commercial kits - vials for PVA & formalin
• 3 parts fixative to 1 part stool
• Record time collected and placed in
preservative
Preservation continued
• Polyvinyl mercuric chloride (PVA)
– mercury chloride: fixative
– polyvinyl alcohol: resin aids adherence
Preservation
• 5 - 10% formalin - wet mount/concentrate;
cysts, eggs, larvae preserved long time.
• Sodium-acetate-acetic acid formalin (SAF) -
concentration & permanent stains; albumin
helps adherence; good for iron hematoxylin
stain
• Merthiolate-iodine-formalin (MIF)
– preserves protozoan/worms in wet mount
& concentration (NOT permanent stain)
Examination of fecal specimen
• Consistency (aids ID of protozoan)
– Liquid
• 1o motile protozoan trophozoites
• examine within 1/2 hour of passage
• preserve for permanent smear
– Semi-formed
• also some cysts (semi-formed)
Fecal exam continued
• Formed stool
– protozoan cysts
– examine on day of passage
– refrigerate up to 24 hours
– in formalin for concentration procedures
– in PVA for permanent smears
• NOT in incubator - destroys parasites,
increases bacteria
Fecal exam continued
• Color
– Brown: normal
– Dark: possible bleeding upper GI tract
– Fresh blood: possible bleeding lower GI
tract
• Select areas of stool with blood
Microscopic exam
• General
– correct illumination
– ocular micrometer - not interchangeable
after calibration
– scan edges of coverslip
– observe entire slide (tedious)
– use references: pictures, size charts, stained
positive slides
Direct wet mount
• 1o motile protozoans in liquid/soft stools
• PVA preservation not acceptable - cloudy
• Wet prep - small amount of stool in
– saline: detect worm eggs/larvae & refractile
protozoan cysts
– iodine: shows nuclear detail
• Low objective (high if suspicious object), low
light
Concentration
• Parasites in small amount of fluid
• Removes most of debris
• Based on difference in specific gravity
between parasites and concentrating solution
• Protozoan trophozoites do not survive
• Detects protozoan cysts, worm larvae/eggs
Concentration Procedures
• Fecal concentration techniques are used to
concentrate and quantify fecal parasites when
a direct wet mount of a fecal material fails to
reveal the presence of parasitic organisms
• The methods concentrate the parasites using
gravity or centrifugation
• Fecal concentration techniques include:
– The sedimentation method
– The floatation method
Concentration Procedures
• Sedimentation method
– Uses the principle of gravity or centrifugation to
allow for the recovery of all protozoa, eggs and
larvae present
– The sediment preparation contains more fecal
debri
– Example:  the formalin-ether method
Concentration Procedures
• Floatation method
– Separates protozoan cysts and certain helminth
eggs through the use of a liquid with a high
specific gravity
– Provides a clearer preparation without much debri
compared to the sedimentation method
– Examples:
 zinc sulfate floatation technique
 saturated sodium chloride method
Concentration Procedures
• Field survey techniques
– Kato-Katz
 developed for the semi-concentration and
semiquantitative estimation of shistosome eggs in
feces
– Formol detergent
 is more sensitive than the Kato-Katz technique
because more feces is used
 uses formalin which kills fecal pathogens
Sedimentation
• Easiest procedure
• Centrifugation or gravity - recovers all
organisms and stages
• Uses formalin-ethyl acetate
Flotation
• Yields less fecal debris
• Organisms suspended in zinc sulfate (S.G. =
1.180)
• Recovers most parasites in surface film
• Opens/collapses operculated eggs
• Distorts protozoan cysts
• Heavy eggs may be missed - sink to bottom
• Examine soon for maximum recovery
Other specimens examined for
intestinal parasites
• Duodenal aspirates - e.g., Strongyloides sp.,
Giardia lamblia
• Sigmoidoscopy specimens - e.g., ameba,
Cryptosporidium
• Urine - Schistosoma hematobium & Enterobius
vermicularis ova
• Cellophane tape-Enterobius vermicularis ova
Blood specimen
Parasites that may be detected in blood
specimens include the agents of malaria
(Plasmodium spp.), babesiosis (Babesia spp.),
trypanosomiasis (Trypanosoma spp.),
leishmaniasis (Leishmania donovani), and
filariasis (Wuchereria bancrofti, Brugia malayi,
Loa loa, and Mansonella spp.).
Blood specimen
• The most important techniques to be
performed in the clinical laboratory to assist in
the diagnosis of blood parasites include
preparation, staining, and examination of
thick and thin blood films. Other techniques
used less frequently include the buffy coat
smear and various concentration techniques
reserved for recovery of microfilariae
General laboratory techniques
1. Parasitic (morphological) diagnosis
– Macroscopic
– Microscopic
2. Immunological diagnosis
– Antibody detection
– Antigen detection
3. Molecular diagnosis
4. Culture
5. Animal inoculation
6. Xenodiagnosis
1-Parasitic (morphological) diagnosis

• Laboratory procedures detect organisms within


clinical specimens using morphological criteria,
A. Macroscopic-using our naked eye
• Eg: Stool specimen can be examined with the
naked eye for presence of some adult worms
– E.g. Ascaris, Taenia species, E.vermicularis
and gravid Taenia species
B. Microscopic
• the majority of intestinal, blood, urinary and skin
parasites are usually detected microscopically
• Use different body specimen such as blood, stool,
urine, CSF etc
– either directly or following concentration
techniques
– in stained or unstained preparation
Types of stain
Temporary stain
• Eosin
• Thomson’s stain
• Lugol’s iodine solution
• Sargeant’s stain
• Burrow’s stain
• Acridine orrange
Permanent stain
• Giemsa/field stain
• Trichrome stain
• Iron-hematxiline stain
• Modified ziehl-neelson stain
• Phenol-auromine method
2-Immunodiagnosis:-

• Is based on the detection of :


A. Antibody detection person's serum
• Ab is produced in response to a particular parasitic
infection.
• The Ab. may persist for a long period of time in the
serum after an infection has ended
• antibody tests are unable to distinguish between past
or present infection
B. Antigen detection
– Ag. is excreted by parasites and can be found in
the serum, urine, CSF, feces or other specimens.
– Antigen tests provide evidence of present
infection
Immunodiagnostic techniques are required

• Parasites live in the tissue of internal organ and can


not easily obtained for examination.
• Parasites can be found in specimens only in certain
stages of infection,
– e.g., in the acute stage not in the chronic stage.
• Parasites are present intermittently or in too few
numbers to be easily detected in the specimens.
• The techniques used to detect parasites are complex
or time consuming.
Immunodiagnosis is of particular value for:

• South American trypanosomiasis , Chronic stage


• African trypanosomiasis, when parasitaemia is low
• Leishmaniasis
• Filariasis
• Amoebic liver abscess
• Trichinosis
• Toxoplasmosis
• Toxocarisis
• Hydatid disease
• Schistosomiasis
• Malaria